Figure 4

Pathological examination of the transplanted NOD-IPCs. (a) H&E-stained transplanted NOD-IPCs were examined under the kidney capsule. RCP μ-pieces were identified at the indicated time points (yellow arrowheads). Upper panels. Original magnification, × 100. Scale bar, 200 μm. Lower panels. Original magnification, × 400. Scale bar, 50 μm. (b–d) Immunohistochemical staining of the NOD-IPCs. These cells showed substantial staining by the anti-insulin and anti-PDX-1 antibodies on Day 30; however, they were barely stained on Day 60. The expression of PD-L1 was maintained in the transplanted NOD-IPCs on Day 30; however, the expression was significantly lower on Day 60. Original magnification, × 400. Scale bar, 50 μm. (e, f) On Day 30, small numbers of CD4⁺ and CD8⁺ T cells were observed around the transplanted NOD-IPCs. However, many CD4⁺ and CD8⁺ T cells had accumulated around the transplanted NOD-IPCs and infiltrated deeply into the NOD-IPCs clusters by Day 60. The numbers of CD4⁺ and CD8⁺ T cells were significantly higher on Day 60 than on Day 30. Original magnification, × 400. Scale bar, 25 μm. The lines in the boxes show the median values (*P < 0.05, Mann–Whitney U test). (g–i) The expression of ICA and ZnT8, which were expressed before transplantation, was maintained after transplantation (no difference in staining intensity between the time points). Conversely, GAD65, which was not expressed in vitro, was expressed in NOD-IPCs after transplantation. Moreover, the staining intensity was significantly higher on Day 60 than on Day 30. Original magnification, × 400. Scale bar, 50 μm. The lines in the boxes show the median values (*P < 0.05, Mann–Whitney U test).